Intermediate-sized filaments (IFs) are stable cytoplasmic protein polymers in which the constituent polypeptides interact, intimately and specifically, at various levels of structural hierarchy (for reviews, see Weber & Geisler, In Cancer Cells. The Transformed Phenotype (Levine et al., eds.) 1:169-176, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1984); Quinlan et al., Cell 42:411-419 (1985)). At the first level, the .alpha.-helical parts of IF polypeptides form two-stranded coiled-coils. These dimers closely associate further into 2 to 3-nm rod-like particles comprising pairs of coiled-coils that represent the fundamental tetrameric subunits of IFs. These tetrameric rod particles then may assemble, via 8 to 12-nm annular and/or 2 to 3-nm long protofilamentous intermediates, into the 8 to 12-nm IFs. These various discrete subunit states have been identified, by biochemical and electron microscope techniques, as the result of limited IF disassembly or as intermediates during reassembly of IFs in vitro (Schlaepfer, J. Cell Biol. 74:226-240 (1977); Schlaepfer, J. Ultrastruct. Res. 61:149-157 (1977); Ahmadi et al., In Fibrous Proteins: Scientific, Industrial, and Medical Aspects (Parry, D., et al., eds.) 2, pp. 161-166 (1980); Renner et al., J. Mol. Biol. 149:285-306 (1981); Steinert et al., In Electron Microscopy of Proteins (Harris, J. ed.) 1, pp. 125-166, Academic Press, N.Y. (1981); Woods & Gruen; Aust. J. Biol. Sci. 34:515-526 (1981); Franke et al., Biol. Cell 46:257-268 (1982); Geisler & Weber, EMBO J. 1:1649-1656 (1982); Geisler et al., J. Mol. Biol. 182:173-177 (1985); Aebi et al., J. Cell Biol. 97:1131-1143 (1983); Quinlan et al., J. Mol. Biol. 178:365-388 (1984); Quinlan et al., J. Mol. Biol. 192:337-349 (1986); Sauk et al., J. Cell Biol. 99:1590-1597 (1984); Eichner et al., In Intermediate Filaments (Wang, E. et al., eds.), Ann. N.Y. Acad. Sci. 455:381-402 (1985); Ip et al., J. Mol. Biol. 183:365-375 (1985); Ip et al., In Intermediate Filaments (Wang, et al., eds.), Ann. N.Y. Acad. Sci. 455:185-199 (1985)).
A special requirement for polypeptide chain interaction exists in the cytokeratins, which are obligatory heteropolymers (Lee & Baden, Nature (London) 264:377-379 (1976); Steinert et al., J. Mol. Biol. 108:547-567 (1976); Milestone, J. Cell Biol. 88:317-332 (1981); Hatzfeld & Franke, J. Cell Biol. 101:1826-1841 (1985); Eichner et al., J. Cell Biol. 102:1767-1777 (1986)) formed by tetramers containing two chains of representatives of either cytokeratin subfamily, i.e., the basic (type II) and acidic (type I) cytokeratins (see, for example, Crewther et al., Int. J. Biol. Macromol. 5:267-274 (1983); Franke et al., Proc. Natl. Acad. Sci. (USA) 80:7113-7117 (1983); Quinlan et al., J. Mol. Biol. 178:365-388 (1984); Sun et al., In Cancer Cells. The Transformed Phenotype (Levine et al., eds.), 1, pp. 169-176, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1984); Woods & Inglis, Int. J. Biol. Macromol. 6:277-283 (1984); Fuchs, E. et al., In Intermediate Filaments (Wang, E. et al., eds.), Ann. N.Y. Acad Sci. 455:436-450 (1985). It is not yet clear whether the coiled-coil polypeptide dimers are homotypic or heterotypic (Franke et al., Proc. Natl. Acad. Sci. (USA) 80:7113-7117 (1983); Gruen & Woods, Biochem. J. 209:587-598 (1983); Quinlan et al., J. Mol. Biol. 178:365-388 (1984); Parry et al., Biochem. Biophys. Res. Commun. 127:1012-1018 (1985); Ward et al., Biochemistry 24:4429-4434 (1985)). It is evident, however, that the type I and type II polypeptides present in the subunits are held together, in large part, by strong hydrogen bonds. Increasing concentrations of urea reveal a relatively sharp dissociation curve characteristic of a given cytokeratin combination (Franke et al., Proc. Natl. Acad. Sci. (USA) 80:7113-7117 (1983)).
IF proteins are members of a large multigene protein family that share amino acid sequence homologies (Geisler & Weber, Proc. Natl. Acad. Sci. (USA) 78:4120-4123 (1998); Geisler & Weber, EMBO J. 1:1649-1656 (1982); Quax et al., Cell 35:215-223 (1983)). Amino acid sequence data as well as biochemical studies have revealed a common structural three-domain organization for all types of IF proteins: (1) a non-helical head domain variable in length and amino acid sequence; (2) a central highly .alpha.-helical rod domain of.about.310 amino acids, and (3) a non-helical tail domain of variable length, which, at least in its first part, is not .alpha.-helical (Steinert, et al., supra). The rod domain, which contains most of the IF protein sequence homologies, can be subdivided into three distinct coiled-coil domains, designated coils 1a, 1b and 2 (Geisler & Weber, EMBO J. 1:1649-1656 (1982); Weber & Geisler, In Cancer Cells. The Transformed Phenotype (Levine et al., eds.) 1, pp. 153-159, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1984)), which are separated by short non-.alpha.-helical spacers ("linkers").
On the basis of the relative degree of homology of the rod domain sequences, the IF proteins can be divided into three major classes (Weber & Geisler, supra; Steinert et al., Cell 42:411-419 (1985)) that share not more than 30% homology with each other. One group (type III IF proteins) comprises the non-epithelial proteins desmin, vimentin, glial filament protein and the neurofilament proteins NF-L, NF-M and NF-H, exibiting .gtoreq.50% sequence homology within this group. The two other groups are the acidic (type I) and the basic (type II) cytokeratins.
Studies involving limited proteolysis of IF subunits indicate that the rod domain represents the structural building block and that isolated rod fragments alone do not assemble into long protofilaments and IFs (Steinert, J. Mol. Biol. 123:49-70 (1978); Geisler et al., Cell 30:277-286 (1982)). For non-keratinous IF proteins, it has been reported that enzymic removal of the head piece, or parts thereof, results in assembly-incompetent fragments (Traub & Vorgias, J. Cell. Sci. 63:43-67 (1983); Kaufmann et al., J. Mol. Biol. 185:733-742 (1985); see, however, Lu & Johnson, Int. J. Biol. Macromol. 5:347-350 (1983)). Experiments in which epidermal cytokeratins have been treated with chymotrypsin (Sauk et al., J. Cell-Biol. 99:1590-1597 (1984)) also suggested that removal of both heads and tails of cytokeratins leads to fragments that have lost their capability to form IF. From the finding that the shortest IF polypeptide of known sequence, cytokeratin 19, is devoid of a non-.alpha.-helical tail region (Bader et al., EMBO J. 5:1865-1875 (1986)) but assembles into IFs when combined with type II cytokeratins (Hatzfeld & Franke, J. Cell Biol. 101:1826-1841 (1985)), it has been concluded that the head piece but not the tail is essential for IF assembly. This conclusion is in agreement with the result of Kaufmann et al., supra, who found that proteolytic removal of the last 27 amino acids from the tail of desmin leaves a molecule that can still form IF.
More controversial results have been reported on the structure of the rod domain of cytokeratins. Upon tryptic digestion of bovine epidermal cytokeratins, .alpha.-helical particles of M.sub.r 42,000 and 108,000 were found that were originally interpreted to be polypeptide trimers (see, for example, Skerrow et al., J. Biol. Chem. 248:4820-4826 (1973)) but were then recognized as tetramers (Woods & Gruen, Aust. J. Biol. Sci. 34:515-526 (1981); Gruen & Woods, Biochem. J. 209:587-598 (1983); Woods, Biochem. Int. 7:769-774 (1983)).
More recently, particles containing tryptic fragments from murine epidermal cytokeratin IFs were resolved into two fractions. These fractions were interpreted to be tetramers and dimers by Parry et al. (Biochem. Biophys. Res. Commun. 127:1012-1018 (1985)). These authors concluded that the dimeric particle represented heterotypic coiled-coils arranged in parallel and in register. While these data suggest that both type I and type II cytokeratins, at least those of sheep wool and mammalian epidermis, are recovered in the same proteolytically obtained rod fragments, it is not clear whether the association of the complementary cytokeratins is directed by elements located in the rod domain, or whether certain peptides involved in the recognition and alignment of these chains are located outside of the rod domain. For example, it has been discussed that the head and tail domains of the cytokeratins might be responsible for the complementarity of binding and the strong hydrogen bonds formed between type I and type II cytokeratins (Weber & Geisler, supra).
During the development of certain organs, the organization of one-layered polar epithelia changes and transforms into stratified epithelia. At this point, the induction of cytokeratin synthesis (i.e., cytokeratins 3-6 and 9-17) appears to be related to the stratification process. Two of the earliest stratification-related cytokeratin polypeptides are cytokeratin 4, a representative of the type II subfamily, and the type I cytokeratin 13, which are expressed, at least transiently, during the development of all diverse stratified epithelia studied so far, including embryonic epidermis. While these two cytokeratins seem to disappear in later maturation stages of epidermis, they represent the most abundant cytokeratins in several adult non-epidermal stratified epithelia such as oral and lingual mucosa, laryngeal and pharyngeal epithelia, epiglottis, esophagus, exocervix and vagina. See Banks-Schlegel, J. Ceil Biol. 93:551-559 (1982); Banks-Schlegel, Cancer Res. 44:1153-1157 (1984); Fuchs, E., et al., In Intermediate Filaments (Wang, E., et al., eds.), Ann. N.Y. Acad. Sci. 455:436-450 (1985); Moll, R., et al., Cell 31:11-24 (.1982); Moll et al., Differentiation 23:256-269 (1983); Moll R., et al., Lab. Invest. 49:599-610 (1983); Nagle, R. B., et al., Differentiation 30:130-140 (1985); Ouhayoun, J.-P., et al., Differentiation 30:123-129 (1985); Quinlan, R. A. et al., In Intermediate Filaments (Wang, E. et al., eds.), Ann. N.Y. Acad. Sci. 455:282-306 (1985).
Corresponding cytokeratin polypeptides abundant in, for example, esophageal epithelium, have been described in several animal species. Cooper, D., et al., J. Biol. Chem. 261:4646-4654 (1986); Cooper et al., Lab. Invest. 52:243-256 (1985); Doran, T. I., et al., Cell 22:17-25 (1980); Franke, W. W., et al., J. Mol. Biol. 153:933-959 (1981); Knapp, B., et al., J. Biol. Chem. 262:938-945 (1987); Milestone, L. M., J. Cell. Biol. 88:317-322 (1981); Schiller, D. L:, et al., EMBO J. 1:761-769 (1982). Consequently, the "expression pair" of cytokeratins 4 and 13 has been regarded as the cytoskeletal hallmark for epithelial differentiation of the "esophageal type".
The histodiagnostic detection of intermediate filament proteins with specific antibodies is known. Such antibodies may be used to determine whether a given tumor growth is of epithelial origin. See, for example, Bannasch, P., et al., Proc. Natl. Acad. Sci (USA) 77:4948-4952 (1980). Moreover, antibodies may be used to detect intermediate filament proteins in metastases or other tissue lesions for the determination of the tissue of origin of the primary tumor.
The composition of the desmosome plaque has been especially thoroughly investigated, the main proteins of which, desmoglein, plakoglobin and desmoplakins I and II are histodiagnostically typical for epithelia and epithelial tumors as well as for some other tissues such as myocardiac tissue, meninges and meningiomas. The appearance of fragments of these main proteins in body fluids may be used as an indicator for cell lesions or destructive processes in epithelial and meningeal tissue. Thus, it is desirable to be able to detect such proteins in a body fluid.
Histological identification of tumor associated tissue is limited by the accessibility of the tissue, and by the fact that many tissue samples are not identifiable by histological means. Thus, a need exists for diagnostic tests for the detection and characterization of proteins characteristic of cell types from both tissue and body fluids.